Hyaluronic acid is a natural mucopolysaccharide present at varying concentrations in practically all tissues. As any expert in the art knows, aqueous solutions of hyaluronic acid or of the salts or derivatives thereof, or of polysaccharides in general, are characterized by notable viscosity, slipperiness, and ability to reduce friction, a characteristic which is at the basis of the presence and function of polysaccharides of the same family as hyaluronic acid in the bodies of humans and other animals (Michels R. G. et al., "Sodium hyaluronate in anterior and posterior segment surgery". Physicochemical and Pharmacological Characteristics of Hyaluronic Acid, 1-15, 1989)
Because of these qualities, polysaccharides of the same family as hyaluronic acid (both natural polysaccharides and those obtained by chemical synthesis on natural compounds) have been widely researched. In particular, great effort has been put into identifying methods by which thin layers of hyaluronic acid (Hyalectin fraction, as described in European patent No. 0138572) or the derivatives thereof (U.S. Pat. No. 4,851,521) can be permanently fixed to the surface of other materials. The aim of this research was to create objects with improved surface properties, while maintaining the overall characteristics of the material of which they are made (said material will hereafter be referred to as the substrate). In particular, because of their high degree of hydrophilia, hyaluronic acid and the derivatives thereof are especially suitable for making objects whose use requires that their surfaces resist adhesion to the cell species present in the tissues or biological fluids. Such surfaces are of particular interest in applications wherein adhesion between materials and cells can cause damage to biological tissues (Kaufman, H. E. et al., Science, 189, 525, 1977).
Modification of the surfaces of materials with hyaluronic acid or the derivatives thereof has proved difficult for many researchers. One of the first things one notices is that hyaluronic acid solutions have a rather high surface tension, the same as or slightly less than that of water (F. H. Silver et al., Journal of Applied Biomaterials, 5, 89, 1994). It is well known that to obtain a homogeneous coating by the application of a solution, the applied material must have a surface tension which is lower than that of the substrate in order to obtain complete, even coverage. Moreover, almost all polymer materials which can be used as substrates present a surface tension which is lower than that of water, a characteristic which prevents the formation of a thin layer of hyaluronic acid covering the substrate evenly (Garbassi F. et al., "Polymer Surfaces, from Physics to Technology", Wiley, Chichester, 304, 1994).
It should be noted that hyaluronic acid is water soluble, so any objects obtained by simply coating them with a layer of hyaluronic acid solution instantly lose their coating on contact with aqueous solutions, including biological fluids. Hyaluronic acid derivatives, even those which are not water soluble, are in any case extremely hydrophilic and have a strong tendency to swell in the presence of water or aqueous solutions (H. N. Joshi and E. M. Topp, International Journ. of Pharm. 80 (1992) 213-225). In aqueous environments, this characteristic rapidly causes the detachment of the hydrophilic surface layer applied to the substrate by a simple coating process using a solution. For these reasons, methods involving a chemical bond between the substrate surface and hyaluronic acid or its derivatives have been studied.
The presence of a stable chemical bond prevents the surface layer from being dissolved and lends stronger, longer-lasting surface properties to the object. The realization of a chemical bond between the substrate and the surface layer requires the presence of suitable chemical groups in both. While the chemical structure of hyaluronic acid ensures the presence of various suitable functions, the surface of most synthetic materials is not particularly suitable for this type of operation. For this reason, processes for the creation of a chemical bond between a surface layer of hyaluronic acid or its derivatives and a synthetic substrate usually consist of two steps. In the first step suitable chemical groups are introduced onto the surface, then in the second step, a reaction is induced between the chemical groups introduced onto the substrate surface and hyaluronic acid or its derivatives. For example, U.S. Pat. Nos. 4,657,820, 4,663,233, 4,722,867, 4,801,475, 4,810,586, 4,959,074, 5,023,114 and 5,037,677 describe the use of an intermediate layer between the substrate and the hyaluronic acid coating. This intermediate layer physically adheres to the substrate and contains chemical groups which are suitable for the formation of a bond with the chemical groups of hyaluronic acid. To facilitate spreading and ensure even coating of the substrate by the hyaluronic acid, the aforesaid patents also describe the use of albumin which, when added to hyaluronic acid, improves its ability to dampen the intermediate layer evenly.
Other documents describe the use of plasma technology to introduce reactive groups onto the substrate. This technique (Garbassi F. et al., "Polymer Surfaces, from Physics to Technology", Wiley, Chichester, 6, 1994), makes it possible to modify the surface of polymer materials in a fast, effective manner. For instance, international patent application No. WO 94/06485, describes the introduction of functional groups onto the surface of a polymeric material by treatment with methanol plasma. The treated material is then placed in contact with an epihydrochlorine solution which guarantees the presence of groups suited to reaction with polysaccharides.
Other articles (Acta Physiologica Scandinava, 116, 201, 1982; Journal of Biomedical Materials Research, 18, 953, 1984, Elan et al.) describe a treatment with oxygen plasma, followed by the application of 3-glycidoxypropyltrimethoxy silane. Surfaces thus treated are used for the formation of covalent bonds with polysaccharides.
Although the above described methods are generally satisfactory, they nonetheless each present some difficulties. In particular, the use of an intermediate layer calls for its composition to be adapted to the nature of the substrate, so as to enhance adhesion as much as possible. In the case of the production of objects constituted by new materials, or materials which are rarely used, much time and effort is taken up in identifying the most suitable formulation for the intermediate layer. If the objects to be coated are composed of different materials, it is difficult to apply a suitable intermediate layer for each component while avoiding overlapping and protrusion of the intermediate layers in unsuitable places. Moreover, it may be undesirable to use albumin to enhance the wettability of the substrate, especially in the case of products intended for biomedical applications.
Regarding the other examples cited, it is preferable to avoid using epihydrochlorine and 3-glycidoxypropyltrimethoxy silane, as these two compounds are known to be major health hazards. Indeed, according to the classification of dangerous substances issued by the European Union, these compounds are coded as "R45" and "R40" respectively, signifying a health risk, as reported in most catalogues for chemical products and reagents. This designation indicates, in the first case, that the product can cause cancer, and in the second case that there is a risk of irreversible effects.
More generally, the total number of reactions which involve functional groups immobilized on a surface and large molecules, such as polysaccharides, is seriously limited by the effect commonly known as steric hindrance. The large size of the polysaccharide molecule prevents or impedes contact between reactive groups so that the probability of an effective reactive encounter is decidedly low.
Other methods described in the art involve the reaction between polysaccharides and amino groups. Japanese patent JP 04126074 (Apr. 27, 1992) describes the use of treatment with ammonia plasma to introduce amino groups on the surface of polymer substrates. The amino groups are then reacted with hyaluronic acid or other polysaccharides by the use of a condensing agent. In U.S. Pat. No. 4,810,784, the surface of an object made of polymer material is treated with reactive solutions, so as to introduce negative electrostatic charges onto the surface itself. The surface thus treated is placed in contact with an aqueous solution of polyethylene imine (PEI), a polymer characterized by the presence of amino groups and a positive electrostatic charge. The interaction between the different charges binds PEI to the modified surface, to produce a surface rich in amino groups. Heparin and other polysaccharides are bound to the aminated surface after treatment with nitrite solutions. It is a known fact in organic chemistry that the action of nitrites causes the formation of aldehyde groups. These react with the aminated surface, binding the polysaccharide irreversibly to the surface itself. The same reaction is used when aldehyde groups are introduced by bland oxidation with periodate (C. Brink et al., "Colloids and Surfaces", 149, 66, 1992).
The reaction between PEI and any aldehyde groups present or introduced on the polysaccharide is, moreover, sometimes used to bind the polysaccharide, in various conformations, to the surface of the object (E. Ostenberg et al., Journal of Biomedical Materials Research, 29, 741, 1995). U.S. Pat. No. 5,409,696 describes the modification of the surface of materials by treatment with plasma containing water vapor and the subsequent reaction of the treated surface with PEI. The surface thus obtained is rich in amino groups and is able to bind heparin and other polysaccharides irreversibly by the action of condensing agents. Typically, the reaction between carboxy groups of the polysaccharide and amino groups of the surface is promoted by ethyldimethylaminopropyl-carbodiimide (EDC). The use of this process to coat the insides of tubes intended to come into contact with the blood has been described by P. V. Narayanan (Journal of Biomaterials Science, Polymer Edition, 6, 181, 1994).
Research has shown that the processes described in the cited patents and articles are not entirely satisfactory as far as the making of objects with surfaces modified by hyaluronic acid or its derivatives is concerned. Indeed, the introduction of amino-type functional groups by means of ammonia plasma, as described in Patent No. JP 04126074 (Apr. 27, 1992), is not very practical in a production process. Experts in the field know that the density of the functional groups introduced by this technique onto the surface of the substrate is rather low, and depends too much on the precise geometry of the reactor used for the plasma treatment, on the nature of the substrate, on the presence of additives and/or contaminants on the surface of and inside the substrate and on the storage conditions of the substrate before and after treatment. For this reason the technique is difficult to apply to industrial production. This negative aspect is recognized by those working in the field and, in the above-noted U.S. Pat. Nos. 4,810,874 and 5,409,696, it is counteracted by using PEI, which allows a high density of amino groups to be obtained. Although these last processes effectively solve the problems involved in the first stage of the process, that is the introduction of reactive groups on the surface of the material, they are not so effective in the second stage, which involves binding hyaluronic acid or derivatives thereof to the surface. Indeed, as we said previously, U.S. Pat. No. 4,810,874 recommends the activation of heparin or other polysaccharides by chemical treatment. It is not, therefore, possible to use the polysaccharide as such, but it is necessary to first modify it by a chemical operation, incurring extra costs in terms of time, reagents, labor and refuse disposal. Moreover, unlike other polysaccharides, hyaluronic acid is only slightly sensitive to the partial oxidation reactions which allow reactive aldehyde-type groups to be introduced on the polysaccharide (J. E. Scott and M. J. Tigwell, Biochem. J., 173, 103, 1978; B. J. Kvam et al., Carbohydrate Research, 230, 1, 1992). As far as U.S. Pat. No. 5,409,696 is concerned, when the process proposed therein is carried out, it does not produce a surface structure able to exploit to the fullest extent the intrinsic characteristics of hyaluronic acid. When the process described in U.S. Pat. No. 5,409,696 is used, on the other hand, as shown in the comparative testing set forth herein, it is not possible to obtain surface structures able to inhibit cell adhesion. Similar results are observed when, instead of hyaluronic acid itself, its water-soluble semisynthetic esters are used (EPA 0216453). Evidently, when this process is used, the way in which a bond is formed between the aminated surface and polysaccharide does not allow the hydrophilic characteristics of hyaluronic acid or its derivatives to be exploited to the fullest extent.
It must not be overlooked that the process which is the subject of U.S. Pat. No. 5,409,696 can be applied only in the surface modification of polymer materials, as indicated by its title "Radio frequency plasma treated polymeric surfaces having immobilized antithrombogenic agents" and by the operational instructions thereof. In common biomedical and surgical practice, ceramic or metallic materials are frequently used, so it is hoped that the modification processes can be applied to such substrates too. This description demonstrates that a method must be devised whereby a chemical bond can be formed, simply and reliably, between substrates of any nature and hyaluronic acid or its derivatives, in such a way that the intrinsic characteristics thereof can be exploited to the fullest extent possible.